EP2124999A2 - Antagonistes de l'activine-actrii et ses utilisations pour accroître les niveaux de globules rouges - Google Patents

Antagonistes de l'activine-actrii et ses utilisations pour accroître les niveaux de globules rouges

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Publication number
EP2124999A2
EP2124999A2 EP07863068A EP07863068A EP2124999A2 EP 2124999 A2 EP2124999 A2 EP 2124999A2 EP 07863068 A EP07863068 A EP 07863068A EP 07863068 A EP07863068 A EP 07863068A EP 2124999 A2 EP2124999 A2 EP 2124999A2
Authority
EP
European Patent Office
Prior art keywords
amino acid
polypeptide
actrii
activin
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP07863068A
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German (de)
English (en)
Other versions
EP2124999B1 (fr
Inventor
Matthew L. Sherman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Acceleron Pharma Inc
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Acceleron Pharma Inc
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Filing date
Publication date
Priority to EP17182870.0A priority Critical patent/EP3320911B1/fr
Priority to PL11195130T priority patent/PL2468290T3/pl
Application filed by Acceleron Pharma Inc filed Critical Acceleron Pharma Inc
Priority to EP11195130.7A priority patent/EP2468290B1/fr
Priority to EP11195140.6A priority patent/EP2468291B1/fr
Priority to PL07863068T priority patent/PL2124999T3/pl
Priority to EP21205033.0A priority patent/EP4026553A1/fr
Priority to SI200731110T priority patent/SI2124999T1/sl
Priority to DK11195130.7T priority patent/DK2468290T3/en
Priority to MEP-2016-200A priority patent/ME02335B/fr
Priority to EP11195282A priority patent/EP2446896A1/fr
Publication of EP2124999A2 publication Critical patent/EP2124999A2/fr
Application granted granted Critical
Publication of EP2124999B1 publication Critical patent/EP2124999B1/fr
Priority to CY20121101136T priority patent/CY1114827T1/el
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1796—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the mature red blood cell, or erythrocyte, is responsible for oxygen transport in the circulatory systems of vertebrates.
  • Red blood cells carry high concentrations of hemoglobin, a protein that binds oxygen in the lungs at relatively high partial pressure of oxygen (p ⁇ 2 ) and delivers oxygen to areas of the body with a relatively low p ⁇ 2 .
  • Mature red blood cells are produced from pluripotent hematopoietic stem cells in a process termed erythropoiesis. In post-natal individuals, erythropoiesis occurs primarily in the bone marrow and in the red pulp of the spleen. The coordinated action of various signaling pathways control the balance of cell proliferation, differentiation, survival and death. Under normal conditions, red blood cells are produced at a rate that maintains a constant red cell mass in the body, and production may increase or decrease in response to various stimuli, including increased or decreased oxygen tension or tissue demand. The process of erythropoiesis begins with the formation of lineage committed precursor cells and proceeds through a series of distinct precursor cell types.
  • the final stages of erythropoiesis occur as reticulocytes are released into the bloodstream and lose their mitochondria and ribosomes while assuming the morphology of mature red blood cell.
  • An elevated level of reticulocytes, or an elevated reticulocyte:erythrocyte ratio, in the blood is indicative of increased red blood cell production rates.
  • Erythropoietin is widely recognized as the most significant positive regulator of erythropoiesis in post-natal vertebrates. Epo regulates the compensatory erythropoietic response to reduced tissue oxygen tension (hypoxia) and low red blood cell levels or low hemoglobin levels. In humans, elevated Epo levels promote red blood cell formation by stimulating the generation of erythroid progenitors in the bone marrow and spleen. In the mouse, Epo enhances erythropoiesis primarily in the spleen.
  • Anemia is a broadly-defined condition characterized by lower than normal levels of hemoglobin or red blood cells in the blood.
  • anemia is caused by a primary disorder in the production or survival of red blood cells. More commonly, anemia is secondary to diseases of other systems (Weatherall & Provan (2000) Lancet 355, 1 169-1 175).
  • Anemia may result from a reduced rate of production or increased rate of destruction of red blood cells or by loss of red blood cells due to bleeding.
  • Anemia may result from a variety of disorders that include, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis, and bone marrow transplantation.
  • Epo typically causes a rise in hemoglobins by about 1-3 g/dL in healthy humans over a period of weeks. When administered to anemic individuals, this treatment regimen often provides substantial increases in hemoglobin and red blood cell levels and leads to improvements in quality of life and prolonged survival. Epo is not uniformly effective, and many individuals are refractory to even high doses (Horl et al. (2000) Nephrol Dial Transplant 15, 43-50). Over 50% of patients with cancer have an inadequate response to Epo, approximately 10% with end-stage renal disease are hyporesponsive (Glaspy et al. (1997) J Clin Oncol 15, 1218-1234; Demetri et al.
  • the disclosure demonstrates that activin antagonists, as well as ActRIIa and ActRIIb antagonists, can be used to increase red blood cell and hemoglobin levels.
  • a soluble form of ActRIIa acts as an inhibitor of activin and, when administered in vivo, increases red blood cell levels in the blood.
  • a milder effect was observed with a soluble form of ActRIIb, which binds Activin A with lesser affinity than soluble ActRIIa.
  • soluble ActRIIa and ActRIIb may affect red blood cell levels through a mechanism other than activin antagonism, the disclosure nonetheless demonstrates that desirable therapeutic agents may be selected on the basis of activin antagonism or ActRII antagonism or both.
  • activin- ActRII antagonists Such agents are referred to collectively as activin- ActRII antagonists. Therefore, in certain embodiments, the disclosure provides methods for using activin-ActRII antagonists, including, for example, activin-binding ActRIIa polypeptides, activin-binding ActRIIb polypeptides, anti-activin antibodies, anti-ActRIIa antibodies, anti-ActRIIb antibodies, activin-, ActRIIb-, or ActRIIa-targeted small molecules and aptamers, and nucleic acids that decrease expression of activin, ActRIIb, or ActRIIa, to increase red blood cell and hemoglobin levels in patients and to treat disorders associated with low red blood cell or hemoglobin levels in patients in need thereof. As described in U.S. Patent Application Serial No.
  • activin-ActRIIa antagonists can be used to promote bone growth and increase bone density. As described herein, the effects of such antagonists on red blood cell levels are more rapid and occur at lower doses than the effects of such antagonists on bone. Thus, in certain embodiments, the disclosure provides methods for using an activin-ActRIIa antagonist to increase red blood cell or hemoglobin levels without causing a significant increase in bone density. For example, a method may cause less than 3%, 5%, 10% or 15% increase in bone density.
  • This selective effect may be achieved by using, for example, lower doses of activin-ActRIIa antagonist, less frequent doses, or by using an activin-ActRIIa antagonist with a shorter serum half-life at doses and frequencies calculated to provide a lower serum concentration.
  • the disclosure provides polypeptides comprising a soluble, activin- binding ActRII polypeptide that binds to activin.
  • the activin binding polypeptide may be an ActRIIa polypeptide or an ActRIIb polypeptide.
  • ActRII polypeptides may be formulated as a pharmaceutical preparation comprising the activin-binding ActRII polypeptide and a pharmaceutically acceptable carrier.
  • the activin-binding ActRII polypeptide may bind to activin with a K D less than 1 micromolar or less than 100, 10 or 1 nanomolar.
  • the activin-binding ActRII polypeptide selectively binds activin versus GDFl 1 and/or GDF8, and optionally with a K D that is at least 10-fold, 20-fold or 50-fold lower with respect to activin than with respect to GDFl 1 and/or GDF8. While not wishing to be bound to a particular mechanism of action, it is expected that this degree of selectivity for activin inhibition over GDFl 1/GDF8 inhibition accounts for effects on bone or erythropoiesis without a consistently measurable effect on muscle. In many embodiments, an ActRII polypeptide will be selected for causing less than 15%, less than 10% or less than 5% increase in muscle at doses that achieve desirable effects on red blood cell levels.
  • the composition may be at least 95% pure, with respect to other polypeptide components, as assessed by size exclusion chromatography, and optionally, the composition is at least 98% pure.
  • An activin-binding ActRIIa polypeptide for use in such a preparation may be any of those disclosed herein, such as a polypeptide having an amino acid sequence selected from SEQ ID NOs: 2, 3, 7 or 12, or having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selected from SEQ ID NOs: 2, 3, 7, 12 or 13.
  • An activin-binding ActRIIa polypeptide may include a functional fragment of a natural ActRIIa polypeptide, such as one comprising at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs: 1-3 or a sequence of SEQ ID NO: 2, lacking the C-terminal 10 to 15 amino acids (the "tail").
  • An activin-binding ActRIIb polypeptide for use in such a preparation may be any of those disclosed herein, such as a polypeptide having an amino acid sequence selected from SEQ ID NOs: 16, 17, 20, or 21 or having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selected from SEQ ID NOs: 16, 17, 20, or 21.
  • An activin-binding ActRIIb polypeptide may include a functional fragment of a natural ActRIIb polypeptide, such as one comprising at least 10, 20 or 30 amino acids of SEQ ID NOs: 15-17 or a sequence lacking the C-terminal 10 to 15 amino acids (the "tail") such as SEQ ID NO: 17.
  • a soluble, activin-binding ActRII polypeptide may include one or more alterations in the amino acid sequence (e.g., in the ligand-binding domain) relative to a naturally occurring ActRII polypeptide.
  • altered ActRIIa and ActRIIb polypeptides are provided in WO 2006/012627, pp. 59-60 and pp. 55-58, respectively, which is incorporated by reference herein.
  • the alteration in the amino acid sequence may, for example, alter glycosylation of the polypeptide when produced in a mammalian, insect or other eukaryotic cell or alter proteolytic cleavage of the polypeptide relative to the naturally occurring ActRII polypeptide.
  • An activin-binding ActRII polypeptide may be a fusion protein that has, as one domain, an ActRII polypeptide, (e.g., a ligand-binding portion of an ActRIIa or ActRIIb) and one or more additional domains that provide a desirable property, such as improved pharmacokinetics, easier purification, targeting to particular tissues, etc.
  • a domain of a fusion protein may enhance one or more of in vivo stability, in vivo half life, uptake/administration, tissue localization or distribution, formation of protein complexes, multimerization of the fusion protein, and/or purification.
  • an activin-binding ActRII fusion protein may include an immunoglobulin Fc domain (wild-type or mutant) or a serum albumin or other polypeptide portion that provides desirable properties such as improved pharmacokinetics, improved solubility or improved stability.
  • an ActRII-Fc fusion comprises a relatively unstructured linker positioned between the Fc domain and the extracellular ActRII domain. This unstructured linker may correspond to the roughly 15 amino acid unstructured region at the C-terminal end of the extracellular domain of ActRII (the "tail"), or it may be an artificial sequence of 1, 2, 3, 4 or 5 amino acids or a length of between 5 and 15, 20, 30, 50 or more amino acids that are relatively free of secondary structure, or a mixture of both.
  • a linker may be rich in glycine and proline residues and may, for example, contain a single sequence of threonine/serine and glycines or repeating sequences of threonine/serine and glycines (e.g., TG 4 or SG 4 singlets or repeats).
  • a fusion protein may include a purification subsequence, such as an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion.
  • a soluble ActRII polypeptide includes one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent.
  • a pharmaceutical preparation may also include one or more additional compounds such as a compound that is used to treat a bone disorder.
  • a pharmaceutical preparation is substantially pyrogen free.
  • an ActRII protein be expressed in a mammalian cell line that mediates suitably natural glycosylation of the ActRII protein so as to diminish the likelihood of an unfavorable immune response in a patient.
  • Human and CHO cell lines have been used successfully, and it is expected that other common mammalian expression systems will be useful.
  • ActRIIa proteins designated ActRIIa-Fc (a form with a minimal linker between the ActRIIa portion and the Fc portion) have desirable properties, including selective binding to activin versus GDF8 and/or GDFl 1 , high affinity ligand binding and serum half life greater than two weeks in animal models.
  • the invention provides ActRII-Fc polypeptides and pharmaceutical preparations comprising such polypeptides and a pharmaceutically acceptable excipient.
  • the disclosure provides nucleic acids encoding a soluble activin- binding ActRII polypeptide, such as an ActRIIa or ActRIIb polypeptide.
  • An isolated polynucleotide may comprise a coding sequence for a soluble, activin-binding ActRII polypeptide, such as described above.
  • an isolated nucleic acid may include a sequence coding for an extracellular domain (e.g., ligand-binding domain) of an ActRII and a sequence that would code for part or all of the transmembrane domain and/or the cytoplasmic domain of an ActRII, but for a stop codon positioned within the transmembrane domain or the cytoplasmic domain, or positioned between the extracellular domain and the transmembrane domain or cytoplasmic domain.
  • an extracellular domain e.g., ligand-binding domain
  • an isolated polynucleotide may comprise a full-length ActRIIa polynucleotide sequence such as SEQ ID NO: 4 or 5 or a full- length ActRIIb polynucleotide sequence such as SEQ ID NO: 18, or a partially truncated version of ActRIIa or ActRIIb, said isolated polynucleotide further comprising a transcription termination codon at least six hundred nucleotides before the 3 '-terminus or otherwise positioned such that translation of the polynucleotide gives rise to an extracellular domain optionally fused to a truncated portion of a full-length ActRII.
  • a preferred nucleic acid sequence for ActRIIa is SEQ ID NO: 14.
  • Nucleic acids disclosed herein may be operably linked to a promoter for expression, and the disclosure provides cells transformed with such recombinant polynucleotides.
  • the cell is a mammalian cell such as a CHO cell.
  • the disclosure provides methods for making a soluble, activin- binding ActRII polypeptide. Such a method may include expressing any of the nucleic acids (e.g., SEQ ID NO: 4, 5 14, 18, or 19) disclosed herein in a suitable cell, such as a Chinese hamster ovary (CHO) cell.
  • a suitable cell such as a Chinese hamster ovary (CHO) cell.
  • Such a method may comprise: a) culturing a cell under conditions suitable for expression of the soluble ActRII polypeptide, wherein said cell is transformed with a soluble ActRII expression construct; and b) recovering the soluble ActRII polypeptide so expressed.
  • Soluble ActRII polypeptides may be recovered as crude, partially purified or highly purified fractions. Purification may be achieved by a series of purification steps, including, for example, one, two or three or more of the following, in any order: protein A chromatography, anion exchange chromatography (e.g., Q sepharose), hydrophobic interaction chromatography (e.g., phenylsepharose), size exclusion chromatography, and cation exchange chromatography.
  • an activin-ActRII antagonist disclosed herein such as a soluble, activin-binding ActRIIa polypeptide or soluble, activin-binding ActRIIb polypeptide, may be used in a method for promoting red blood cell production or increasing red blood cell levels in a subject.
  • the disclosure provides methods for treating a disorder associated with low red blood cell counts or low hemoglobin levels (e.g., an anemia), or to promote red blood cell production, in patients in need thereof.
  • a method may comprise administering to a subject in need thereof an effective amount of activin-ActRII antagonist.
  • the disclosure provides uses of activin-ActRII antagonists for making a medicament for the treatment of a disorder or condition as described herein.
  • the disclosure provides a method for identifying an agent that stimulates production of red blood cells.
  • the method comprises: a) identifying a test agent that binds to activin or a ligand-binding domain of an ActRII polypeptide; and b) evaluating the effect of the agent on the levels of red blood cells, hemoglobin, and/or red blood cell precursor levels (e.g., reticulocyte levels).
  • Figure 1 shows the purification of ActRIIa-hFc expressed in CHO cells.
  • the protein purifies as a single, well-defined peak as visualized by sizing column (left panel) and Coomassie stained SDS-PAGE (right panel) (left lane: molecular weight standards; right lane: ActRIIa-hFc).
  • Figure 2 shows the binding of ActRIIa-hFc to activin and GDF-1 1 , as measured by BiaCore I M assay.
  • Figure 3 shows the effects of ActRIIa-hFc on red blood cell counts in female non- human primates.
  • Female cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or l mg/kg, 10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21.
  • Figure 3 A shows red blood cell (RBC) counts.
  • Figure 3B shows hemoglobin levels. Statistical significance is relative to baseline for each treatment group. At day 57, two monkeys remained in each group.
  • Figure 4 shows the effects of ActRIIa-hFc on red blood cell counts in male non- human primates.
  • Male cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or lmg/kg, 10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21.
  • Figure 4A shows red blood cell (RBC) counts.
  • Figure 4B shows hemoglobin levels. Statistical significance is relative to baseline for each treatment group. At day 57, two monkeys remained in each group.
  • Figure 5 shows the effects of ActRIIa-hFc on reticulocyte counts in female non- human primates.
  • Cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21.
  • Figure 5 A shows absolute reticulocyte counts.
  • Figure 5B shows the percentage of reticulocytes relative to RBCs. Statistical significance is relative to baseline for each group. At day 57, two monkeys remained in each group.
  • Figure 6 shows the effects of ActRIIa-hFc on reticulocyte counts in female non- human primates. Cynomolgus monkeys (four groups of five monkeys each) were treated with placebo or 1 mg/kg, 10 mg/kg or 30 mg/kg of ActRIIa-hFc on day 0, day 7, day 14 and day 21.
  • Figure 6A shows absolute reticulocyte counts.
  • Figure 6B shows the percentage of reticulocytes relative to RBCs. Statistical significance is relative to baseline for each group. At day 57, two monkeys remained in each group.
  • Figure 7 shows results from the human clinical trial described in Example 5, where the area-under-curve (AUC) and administered dose of ActRIIa-hFc have a linear correlation, regardless of whether ActRIIa-hFc was administered intravenously (IV) or subcutaneously (SC).
  • AUC area-under-curve
  • IV intravenously
  • SC subcutaneously
  • Figure 8 shows a comparison of serum levels of ActRIIa-hFc in patients administered IV or SC.
  • Figure 9 shows bone alkaline phosphatase (BAP) levels in response to different dose levels of ActRIIa-hFc.
  • BAP is a marker for anabolic bone growth.
  • Figure 10 depicts the median change from baseline of hematocrit levels from the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dosage.
  • Figure 1 1 depicts the median change from baseline of hemoglobin levels from the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dosage.
  • Figure 12 depicts the median change from baseline of RBC (red blood cell) count from the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dosage.
  • Figure 13 depicts the median change from baseline of reticulocyte count from the human clinical trial described in Example 5. ActRIIa-hFc was administered intravenously (IV) at the indicated dosage.
  • TGF-beta The transforming growth factor-beta (TGF -beta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a large variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. The family is divided into two general branches: the BMP/GDF and the TGF-beta/Activin/BMPl 0 branches, whose members have diverse, often complementary effects.
  • Activins are dimeric polypeptide growth factors that belong to the TGF-beta superfamily. There are three principal activin forms (A, B, and AB) that are homo/heterodimers of two closely related ⁇ subunits (P A P A , P B P B , and P A P B , respectively).
  • the human genome also encodes an activin C and an activin E, which are primarily expressed in the liver, and heterodimeric forms containing ⁇ c or ⁇ e are also known.
  • activins are unique and multifunctional factors that can stimulate hormone production in ovarian and placental cells, support neuronal cell survival, influence cell-cycle progress positively or negatively depending on cell type, and induce mesodermal differentiation at least in amphibian embryos (DePaolo et al., 1991 , Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997, Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963).
  • erythroid differentiation factor isolated from the stimulated human monocytic leukemic cells was found to be identical to activin A (Murata et al., 1988, PNAS, 85:2434). It has been suggested that activin A promotes erythropoiesis in the bone marrow. In several tissues, activin signaling is antagonized by its related heterodimer, inhibin. For example, during the release of follicle-stimulating hormone (FSH) from the pituitary, activin promotes FSH secretion and synthesis, while inhibin prevents FSH secretion and synthesis.
  • FSH follicle-stimulating hormone
  • Other proteins that may regulate activin bioactivity and/or bind to activin include follistatin (FS), follistatin-related protein (FSRP) and ⁇ 2 -macroglobulin.
  • TGF- ⁇ signals are mediated by heteromeric complexes of type I and type II serine/ threonine kinase receptors, which phosphorylate and activate downstream Smad proteins upon ligand stimulation (Massague, 2000, Nat. Rev. MoI. Cell Biol. 1 :169-178).
  • type I and type II receptors are transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine specificity.
  • Type I receptors are essential for signaling; and type II receptors are required for binding ligands and for expression of type I receptors.
  • Type I and II activin receptors form a stable complex after ligand binding, resulting in phosphorylation of type I receptors by type II receptors.
  • Two related type II receptors ActRII, ActRIIa and ActRIIb, have been identified as the type II receptors for activins (Mathews and Vale, 1991 , Cell 65:973-982; Attisano et al., 1992, Cell 68: 97-108).
  • ActRIIa and ActRIIb can biochemically interact with several other TGF- ⁇ family proteins, including BMP7, Nodal, GDF8, and GDFl 1 (Yamashita et al., 1995, J. Cell Biol.
  • ALK4 is the primary type I receptor for activins, particularly for activin A, and ALK-7 may serve as a receptor for activins as well, particularly for activin B.
  • sActRIIa a soluble ActRIIa polypeptide
  • GDF8 or GDFl 1 TGF-beta family members
  • hematopoiesis is a complex process, regulated by a variety of factors, including erythropoietin, G-CSF and iron homeostasis.
  • the terms “increase red blood cell levels” and “promote red blood cell formation” refer to clinically observable metrics, such as hematocrit, red blood cell counts and hemoglobin measurements, and are intended to be neutral as to the mechanism by which such changes occur.
  • a soluble ActRIIb polypeptide is effective to increase reticulocyte levels in vivo, an effect which, over a longer time period is expected to cause increased hemotocrit levels.
  • Activin-ActRII antagonists include, for example, activin-binding soluble ActRIIa polypeptides, activin-binding soluble ActRIIb polypeptides, antibodies that bind to activin (particularly the activin A or B subunits, also referred to as ⁇ A or ⁇ B) and disrupt ActRIIa and/or ActRIIb binding, antibodies that bind to ActRIIa and disrupt activin binding, antibodies that bind to ActRIIb and disrupt activin binding, non-antibody proteins selected for activin, ActRIIb or ActRIIa binding (see e.g., WO/2002/088171, WO/2006/055689, and WO/2002/032925 for examples of such proteins and methods for design and selection of same), randomized peptides selected for activin, ActRIIb, or ActRIIa binding, often affixed to an Fc domain.
  • activin binders that block the type 1 (e.g., a soluble type I activin receptor) and type 11 (e.g., a soluble type II activin receptor) binding sites, respectively, may be linked together to create a bifunctional binding molecule.
  • Nucleic acid aptamers, small molecules and other agents that inhibit the activin-ActRII signaling axis are included as activin-ActRII antagonists.
  • activin-ActRII antagonist activity including inhibin (i.e., inhibin alpha subunit), although inhibin does not universally antagonize activin in all tissues, follistatin (e.g., follistatin-288 and follistatin-315), FSRP, activin C, alpha(2)-macroglobulin, and an M 108 A (methionine to alanine change at position 108) mutant activin A.
  • inhibin i.e., inhibin alpha subunit
  • follistatin e.g., follistatin-288 and follistatin-315
  • FSRP activin C
  • alpha(2)-macroglobulin e.g., follistatin-288 and follistatin-315
  • M 108 A methionine to alanine change at position 108 mutant activin A.
  • alternative forms of activin particularly those with alterations in the type I receptor binding domain can bind to type II receptor
  • nucleic acids such as antisense molecules, siRNAs or ribozymes that inhibit activin A, B, C or E, or, particularly, ActRIIa or ActRIIb expression, can be used as activin-ActRII antagonists.
  • the activin- ActRII antagonist to be used may exhibit selectivity for inhibiting activin-mediated signaling versus other members of the TGF-beta family, and particularly with respect to GDF8 and GDFI l .
  • “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values.
  • the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5- fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
  • the methods of the invention may include steps of comparing sequences to each other, including wild-type sequence to one or more mutants (sequence variants). Such comparisons typically comprise alignments of polymer sequences, e.g., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few).
  • sequence alignment will introduce a "gap" (typically represented by a dash, or "A") in the polymer sequence not containing the inserted or deleted residue.
  • sequence similarity in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.
  • homologous when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
  • the present invention relates to ActRII polypeptides.
  • ActRII refers to the family of type II activin receptors. This family includes both the activin receptor type Ha and the activin receptor type Hb.
  • the present invention relates to ActRIIa polypeptides.
  • ActRIIa refers to a family of activin receptor type Ha (ActRIIa) proteins from any species and variants derived from such ActRIIa proteins by mutagenesis or other modification. Reference to ActRIIa herein is understood to be a reference to any one of the currently identified forms.
  • Members of the ActRIIa family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.
  • ActRIIa polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ActRIIa family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. See, for example, WO/2006/012627.
  • ActRIIa polypeptides include polypeptides derived from the sequence of any known ActRIIa having a sequence at least about 80% identical to the sequence of an ActRIIa polypeptide, and optionally at least 85%, 90%, 95%, 97%, 99% or greater identity.
  • an ActRIIa_polypeptide of the invention may bind to and inhibit the function of an ActRIIa protein and/or activin.
  • An ActRIIa polypeptide may be selected for activity in promoting red blood cell formation in vivo.
  • Examples of ActRIIa polypeptides include human ActRIIa precursor polypeptide (SEQ ID NO: 1) and soluble human ActRIIa polypeptides (e.g., SEQ ID NOs: 2, 3, 7 and 12).
  • the human ActRIIa precursor protein sequence is as follows:
  • the signal peptide is single underlined; the extracellular domain is in bold and the potential N-linked glycosylation sites are double underlined.
  • the human ActRIIa soluble (extracellular), processed polypeptide sequence is as follows:
  • the nucleic acid sequence encoding human ActRIIa precursor protein is as follows
  • the nucleic acid sequence encoding a human ActRIIa soluble (extracellular) polypeptide is as follows: ATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATT GGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGA CAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGT TCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCT ATGACAGGACTGATTGTGTAGAAAAAAAAAAAAGACAGCCCTGAAGTATATTT TTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCA GAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCAC CC (SEQ ID NO: 5)
  • the present invention relates to ActRIIb polypeptides.
  • ActRIIb refers to a family of activin receptor type lib (ActRIIb) proteins from any species and variants derived from such ActRIIb proteins by mutagenesis or other modification. Reference to ActRIIb herein is understood to be a reference to any one of the currently identified forms.
  • ActRIIb family are generally transmembrane proteins, composed of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.
  • ActRIIb polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ActRIIb family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. See, for example, WO/2006/012627.
  • ActRIIb polypeptides include polypeptides derived from the sequence of any known ActRIIb having a sequence at least about 80% identical to the sequence of an ActRIIb polypeptide, and optionally at least 85%, 90%, 95%, 97%, 99% or greater identity.
  • an ActRIIb . polypeptide of the invention may bind to and inhibit the function of an ActRIIb protein and/or activin.
  • ActRIIb polypeptide may be selected for activity in promoting red blood cell formation in vivo.
  • ActRIIb polypeptides include human ActRIIb precursor polypeptide (SEQ ID NO: 15) and soluble human ActRIIb polypeptides (e.g., SEQ ID NO: 16, 17, 20, and 21).
  • the human ActRIIb precursor protein sequence is as follows: MTAPWV ⁇ LALLWGSLWPGSGRGEAETRECIYYNANWELERTSIQSGLERC
  • the signal peptide is single underlined; the extracellular domain is in bold and the potential N-linked glycosylation sites are in boxes.
  • the human ActRIIb soluble (extracellular), processed polypeptide sequence is as follows:
  • nucleic acid sequence encoding a human ActRIIb precursor protein is as follows: (nucleotides 5-1543 of Genbank entry NM_001 106)
  • the nucleic acid sequence encoding a human ActRIIa soluble (extracellular) polypeptide is as follows: TCTGGGCGTGGGGAGGCTGAGACACGGGAGTGCATCTACTACAACGCCA ACTGGGAGCTGGAGCGCACCAACCAGAGCGGCCTGGAGCGCTGCGAAGG CGAGCAGGACAAGCGGCTGCACTGCTACGCCTCCTGGGCCAACAGCTCT GGCACCATCGAGCTCGTGAAGAAGGGCTGCTGGCTAGATGACTTCAACT GCTACGATAGGCAGGAGTGTGTGGCCACTGAGGAGAACCCCCAGGTGTA CTTCTGCTGCTGTGAAGGCAACTTCTGCAACGAGCGCTTCACTCATTTG CCAGAGGCTGGGGGCCCGGAAGTCACGTACGAGCCACCCCCGACAGCCC CCACC (SEQIDNO: 19)
  • the invention relates to soluble ActRII polypeptides.
  • soluble ActRII polypeptide generally refers to polypeptides comprising an extracellular domain of an ActRIIa or ActRIIb protein.
  • soluble ActRII polypeptide includes any naturally occurring extracellular domain of an ActRIIa or ActRIIb protein as well as any variants thereof (including mutants, fragments and peptidomimetic forms).
  • An activin-binding ActRII polypeptide is one that retains the ability to bind to activin, including, for example, activin AA, AB, BB, or forms that include a C or E subunit.
  • an activin-binding ActRII polypeptide will bind to activin AA with a dissociation constant of 1 nM or less.
  • the extracellular domain of an ActRII protein binds to activin and is generally soluble, and thus can be termed a soluble, activin-binding ActRII polypeptide.
  • Examples of soluble, activin-binding ActRIIa polypeptides include the soluble polypeptides illustrated in SEQ ID NOs: 2, 3, 7, 12 and 13. SEQ ID NO:7 is referred to as ActRIIa-hFc, and is described further in the Examples.
  • soluble, activin-binding ActRIIa polypeptides comprise a signal sequence in addition to the extracellular domain of an ActRIla protein, for example, the honey bee mellitin leader sequence (SEQ ID NO: 8), the tissue plaminogen activator (TPA) leader (SEQ ID NO: 9) or the native ActRIla leader (SEQ ID NO: 10).
  • the ActRIIa-hFc polypeptide illustrated in SEQ ID NO: 13 uses a TPA leader.
  • Examples of soluble, activin-binding ActRIIb polypeptides include the soluble polypeptides illustrated in SEQ ID NOs: 16, 17, 20.
  • Activin-binding ActRIIb polypeptides may also comprise a signal sequence in addition to the extracellular domain of an ActRIIb protein, for example, the honey bee mellitin leader sequence (SEQ ID NO: 8) or the tissue plaminogen activator (TPA) leader (SEQ ID NO: 9).
  • a signal sequence in addition to the extracellular domain of an ActRIIb protein, for example, the honey bee mellitin leader sequence (SEQ ID NO: 8) or the tissue plaminogen activator (TPA) leader (SEQ ID NO: 9).
  • Functionally active fragments of ActRII polypeptides can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding an ActRII polypeptide.
  • fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function as antagonists (inhibitors) of ActRII protein or signaling mediated by activin.
  • a functional variant of the ActRIla polypeptides comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3. In certain cases, the functional variant has an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3.
  • a functional variant of the ActRIIb polypeptides comprises an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 16 or 17. In certain cases, the functional variant has an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 17 or 18.
  • Functional variants may be generated by modifying the structure of an ActRII polypeptide for such purposes as enhancing therapeutic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo). Such modified ActRII polypeptides when selected to retain activin binding, are considered functional equivalents of the naturally-occurring ActRII polypeptides.
  • Modified ActRII polypeptides can also be produced, for instance, by amino acid substitution, deletion, or addition. For instance, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (e.g., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • Whether a change in the amino acid sequence of an ActRII polypeptide results in a functional homolog can be readily determined by assessing the ability of the variant ActRII polypeptide to produce a response in cells in a fashion similar to the wild-type ActRII polypeptide.
  • the present invention contemplates specific mutations of the ActRII polypeptides so as to alter the glycosylation of the polypeptide.
  • Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites.
  • Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine or asparagine-X-serine (where "X" is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes.
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the wild-type ActRII polypeptide (for O- linked glycosylation sites).
  • a variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence.
  • Another means of increasing the number of carbohydrate moieties on an ActRII polypeptide is by chemical or enzymatic coupling of glycosides to the ActRII polypeptide.
  • the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal of one or more carbohydrate moieties present on an ActRII polypeptide may be accomplished chemically and/or enzymatically.
  • Chemical deglycosylation may involve, for example, exposure of the ActRII polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetyl glucosamine or N- acetylgalactosamine), while leaving the amino acid sequence intact.
  • Enzymatic cleavage of carbohydrate moieties on ActRII polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol. 138:350.
  • ActRII polypeptide may be adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide.
  • ActRII proteins for use in humans may be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines are expected to be useful as well.
  • This disclosure further contemplates a method of generating mutants, particularly sets of combinatorial mutants of an ActRII polypeptide, as well as truncation mutants; pools of combinatorial mutants are especially useful for identifying functional variant sequences.
  • the purpose of screening such combinatorial libraries may be to generate, for example, ActRII polypeptide variants which bind to activin or other ligands.
  • a variety of screening assays are provided below, and such assays may be used to evaluate variants.
  • an ActRII polypeptide variant may be screened for ability to bind to an ActRII ligand, to prevent binding of an ActRII ligand to an ActRII polypeptide or to interfere with signaling caused by an ActRII ligand.
  • an ActRII polypeptide or its variants may also be tested in a cell-based or in vivo assay. For example, the effect of an ActRII polypeptide variant on the expression of genes involved in hematopoiesis may be assessed. This may, as needed, be performed in the presence of one or more recombinant ActRII ligand proteins (e.g., activin), and cells may be transfected so as to produce an ActRII polypeptide and/or variants thereof, and optionally, an ActRII ligand. Likewise, an ActRII polypeptide may be administered to a mouse or other animal, and one or more blood measurements, such as an RBC count, hemoglobin, or reticulocyte count may be assessed.
  • ActRII polypeptide may be administered to a mouse or other animal, and one or more blood measurements, such as an RBC count, hemoglobin, or reticulocyte count may be assessed.
  • Combinatorially-derived variants can be generated which have a selective or generally increased potency relative to a naturally occurring ActRII polypeptide.
  • mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding a wild-type ActRII polypeptide.
  • the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in destruction of, or otherwise inactivation of a native ActRII polypeptide.
  • Such variants, and the genes which encode them can be utilized to alter ActRII polypeptide levels by modulating the half-life of the ActRII polypeptides.
  • a short half-life can give rise to more transient biological effects and, when part of an inducible expression system, can allow tighter control of recombinant ActRII polypeptide levels within the cell.
  • mutations may be made in the linker (if any) and/or the Fc portion to alter the half-life of the protein.
  • a combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential ActRII polypeptide sequences.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential ActRII polypeptide nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display).
  • the library of potential homologs can be generated from a degenerate oligonucleotide sequence.
  • Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes can then be ligated into an appropriate vector for expression.
  • the synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al., (1984) Annu. Rev.
  • ActRII polypeptide variants can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Ruf et al., (1994) Biochemistry 33: 1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-1 18; Grodberg et al., (1993) Eur. J. Biochem. 218:597- 601 ; Nagashima et al., (1993) J. Biol. Chem.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of ActRII polypeptides.
  • the most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • Preferred assays include activin binding assays and activin-mediated cell signaling assays.
  • the ActRII polypeptides of the invention may further comprise post-translational modifications in addition to any that are naturally present in the ActRII polypeptides.
  • modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • the modified ActRII polypeptides may contain non-amino acid elements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates. Effects of such non-amino acid elements on the functionality of an ActRII polypeptide may be tested as described herein for other ActRII polypeptide variants.
  • an ActRII polypeptide When an ActRII polypeptide is produced in cells by cleaving a nascent form of the ActRII polypeptide, post-translational processing may also be important for correct folding and/or function of the protein.
  • Different cells such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293 have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the ActRII polypeptides.
  • fusion proteins having at least a portion of the ActRII polypeptides and one or more fusion domains.
  • fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin.
  • a fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography.
  • matrices for affinity chromatography such as glutathione-, amylase-, and nickel- or cobalt- conjugated resins are used.
  • Many of such matrices are available in "kit” form, such as the Pharmacia GST purification system and the QIAexpressTM system (Qiagen) useful with (HIS 6 ) fusion partners.
  • a fusion domain may be selected so as to facilitate detection of the ActRII polypeptides. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as "epitope tags," which are usually short peptide sequences for which a specific antibody is available.
  • fusion domains have a protease cleavage site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation.
  • an ActRII polypeptide is fused with a domain that stabilizes the ActRII polypeptide in vivo (a "stabilizer" domain).
  • stabilizing is meant anything that increases serum half life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains (that confer an additional biological function, such as further stimulation of muscle growth).
  • the present invention provides a fusion protein comprising a soluble extracellular domain of ActRIIa fused to an Fc domain (e.g., SEQ ID NO: 6).
  • THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD (A) VSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG PFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN (A) HYTQKSLSLSPGK*
  • the present invention provides a fusion protein comprising a soluble extracellular domain of ActRIIb fused to an Fc domain (e.g., SEQ ID NO: 21).
  • the Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434.
  • the mutant Fc domain having one or more of these mutations e.g., Asp-265 mutation
  • the mutant Fc domain having one or more of these mutations has increased ability of binding to the MHC class I- related Fc-receptor (FcRN) relative to a wildtype Fc domain.
  • an ActRII polypeptide may be placed C-terminal to a heterologous domain, or, alternatively, a heterologous domain may be placed C-terminal to an ActRII polypeptide.
  • the ActRII polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains.
  • the ActRII polypeptides of the present invention contain one or more modifications that are capable of stabilizing the ActRII polypeptides.
  • Such modifications enhance the in vitro half life of the ActRII polypeptides, enhance circulatory half life of the ActRII polypeptides or reducing proteolytic degradation of the ActRII polypeptides.
  • Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ActRII polypeptide and a stabilizer domain), modifications of a glycosylation site (including, for example, addition of a glycosylation site to an ActRII polypeptide), and modifications of carbohydrate moiety (including, for example, removal of carbohydrate moieties from an ActRII polypeptide).
  • stabilizer domain not only refers to a fusion domain (e.g., Fc) as in the case of fusion proteins, but also includes nonproteinaceous modifications such as a carbohydrate moiety, or nonproteinaceous moiety, such as polyethylene glycol.
  • the present invention makes available isolated and/or purified forms of the ActRII polypeptides, which are isolated from, or otherwise substantially free of, other proteins.
  • ActRII polypeptides will generally be produced by expression from recombinant nucleic acids.
  • the invention provides isolated and/or recombinant nucleic acids encoding any of the ActRII polypeptides (e.g., soluble ActRIIa polypeptides and soluble
  • ActRIIb polypeptides including fragments, functional variants and fusion proteins disclosed herein.
  • SEQ ID NO: 4 encodes the naturally occurring human ActRIIa precursor polypeptide
  • SEQ ID NO: 5 encodes the processed extracellular domain of ActRIIa
  • SEQ ID NO: 18 encodes the naturally occurring human ActRIIb precursor polypeptide
  • SEQ ID NO: 19 encodes the processed extracellular domain of ActRIIb.
  • the subject nucleic acids may be single-stranded or double stranded. Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for making ActRII polypeptides or as direct therapeutic agents (e.g., in a gene therapy approach).
  • the subject nucleic acids encoding ActRIIa polypeptides are further understood to include nucleic acids that are variants of SEQ ID NO: 4 or 5.
  • the subject nucleic acids encoding ActRIIb polypeptides are farther understood to include nucleic acids that are variants of SEQ ID NO: 18 or 19.
  • Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants.
  • the invention provides isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 4, 5, 18, or 19.
  • nucleic acid sequences complementary to SEQ ID NOs: 4, 5, 18, or 19, and variants of SEQ ID NOs: 4, 5, 18, or 19 are also within the scope of this invention.
  • nucleic acid sequences of the invention can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library.
  • nucleic acids of the invention also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ ID NOs: 4, 5, 18, or 19, complement sequence of SEQ ID NOs: 4, 5, 18, or 19, or fragments thereof.
  • appropriate stringency conditions which promote DNA hybridization can be varied.
  • appropriate stringency conditions which promote DNA hybridization can be varied. For example, one could perform the hybridization at 6.0 x sodium chloride/sodium citrate (SSC) at about 45 0 C, followed by a wash of 2.0 x SSC at 50 °C.
  • SSC sodium chloride/sodium citrate
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50 °C to a high stringency of about 0.2 x SSC at 50 0 C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 °C, to high stringency conditions at about 65 0 C. Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed.
  • the invention provides nucleic acids which hybridize under low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature.
  • Isolated nucleic acids which differ from the nucleic acids as set forth in SEQ ID NOs: 4, 5, 18, or 19 due to degeneracy in the genetic code are also within the scope of the invention.
  • a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid sequence of the protein.
  • CAU and CAC are synonyms for histidine
  • nucleotides up to about 3-5% of the nucleotides
  • nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.
  • the recombinant nucleic acids of the invention may be operably linked to one or more regulatory nucleotide sequences in an expression construct.
  • Regulatory nucleotide sequences will generally be appropriate to the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.
  • said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the invention.
  • the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
  • An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.
  • the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding an ActRII polypeptide and operably linked to at least one regulatory sequence.
  • Regulatory sequences are art-recognized and are selected to direct expression of the ActRII polypeptide.
  • the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). For instance, any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding an ActRII polypeptide.
  • Such useful expression control sequences include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda , the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ -mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
  • a recombinant nucleic acid of the invention can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both.
  • Expression vehicles for production of a recombinant ActRII polypeptide include plasmids and other vectors.
  • suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL- derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
  • vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
  • derivatives of viruses such as the bovine papilloma virus (BPV-I), or Epstein- Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-I bovine papilloma virus
  • pHEBo Epstein- Barr virus
  • examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems.
  • the various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A
  • baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the ⁇ -gal containing pBlueBac III).
  • a vector will be designed for production of the subject ActRII polypeptides in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wise).
  • a vector will be designed for production of the subject ActRII polypeptides in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wise).
  • the subject gene constructs can be used to cause expression of the subject ActRII polypeptides in cells propagated in culture, e.g., to produce proteins, including fusion proteins or variant proteins, for purification.
  • This disclosure also pertains to a host cell transfected with a recombinant gene including a coding sequence (e.g., SEQ ID NO: 4, 5, 18, or 19) for one or more of the subject ActRII polypeptides.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • an ActRII polypeptide of the invention may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.
  • the present invention further pertains to methods of producing the subject ActRII polypeptides.
  • a host cell transfected with an expression vector encoding an ActRIIa or ActRIIb polypeptide can be cultured under appropriate conditions to allow expression of the ActRII polypeptide to occur.
  • the ActRII polypeptide may be secreted and isolated from a mixture of cells and medium containing the ActRII polypeptide.
  • the ActRII polypeptide may be retained cytoplasmically or in a membrane fraction and the cells harvested, lysed and the protein isolated.
  • a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
  • the subject ActRIl polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaff ⁇ nity purification with antibodies specific for particular epitopes of the ActRIl polypeptides and affinity purification with an agent that binds to a domain fused to the ActRIl polypeptide (e.g., a protein A column may be used to purify an ActRIIa-Fc or ActRIIb-Fc fusion).
  • the ActRIl polypeptide is a fusion protein containing a domain which facilitates its purification.
  • purification is achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography.
  • the purification could be completed with viral filtration and buffer exchange.
  • ActRIIa-hFc protein was purified to a purity of >98% as determined by size exclusion chromatography and >95% as determined by SDS PAGE. This level of purity was sufficient to achieve desirable results in mice, rats and non- human primates.
  • a fusion gene coding for a purification leader sequence such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant ActRIl polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni 2+ metal resin.
  • the purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified ActRIl polypeptide (e.g., see Hochuli et al., (1987) J. Chromatography 41 1 : 177; and Janknecht et al., PNAS USA 88:8972).
  • fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
  • activin-ActRII signaling can be used to increase red blood cell or hemoglobin levels.
  • soluble ActRIIa and ActRIIb polypeptides and particularly ActRIIa-Fc and ActRIIb-Fc, are preferred antagonists, and although such antagonists may affect red blood cell levels through a mechanism other than activin antagonism (e.g., activin inhibition may be an indicator of the tendency of an agent to inhibit the activities of a spectrum of molecules, including, perhaps, other members of the TGF-beta superfamily, and such collective inhibition may lead to the desired effect on hematopoiesis), other types of activin-ActRII antagonists are expected to be useful, including anti-activin (e.g., activin PA, ⁇ , ⁇ c and ⁇ ) antibodies, anti-ActRIIa antibodies, anti-ActRIIb antibodies, antisense, RNAi or ribozyme nucleic acids that inhibit the production of ActRIIa and/or ActRIIb
  • anti-activin e.g
  • an antibody that is specifically reactive with an ActRII polypeptide e.g., a soluble ActRIIa or ActRIIb polypeptide
  • an antibody that is specifically reactive with an activin ⁇ A, ⁇ , ⁇ c or ⁇ e polypeptide, or any heterodimer thereof, and which disrupts ActRIIa and/or ActRIIb binding may be used as an antagonist.
  • anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the activin, ActRIIa or ActRIIb polypeptide, an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • An immunogenic portion of an ActRII or activin polypeptide can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • antibody-producing cells can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an activin, ActRIIa or ActRIIb polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
  • antibody as used herein is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes fragments or domains of immunoglobulins which are reactive with a selected antigen.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility and/or interaction with a specific epitope of interest.
  • the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein.
  • Non-limiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab' , Fv, and single chain antibodies (scFv) containing a V[L] and/or V[H] domain joined by a peptide linker.
  • the scFv's may be covalently or non- covalently linked to form antibodies having two or more binding sites.
  • the term antibody also includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant antibodies.
  • recombinant antibody means an antibody, or antigen binding domain of an immunoglobulin, expressed from a nucleic acid that has been constructed using the techniques of molecular biology, such as a humanized antibody or a fully human antibody developed from a single chain antibody. Single domain and single chain antibodies are also included within the term “recombinant antibody”.
  • an antibody of the invention is a monoclonal antibody, and in certain embodiments, the invention makes available methods for generating novel antibodies.
  • a method for generating a monoclonal antibody that binds specifically to an ActRlIa polypeptide, ActRIIb polypeptide, or activin polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the antigen polypeptide effective to stimulate a detectable immune response, obtaining antibody- producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody- producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the antigen.
  • antibody- producing cells e.g., cells from the spleen
  • a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to the antigen.
  • the monoclonal antibody may be purified from the cell culture.
  • the adjective "specifically reactive with” as used in reference to an antibody is intended to mean, as is generally understood in the art, that the antibody is sufficiently selective between the antigen of interest (e.g., an activin, ActRIIa or ActRIIb polypeptide) and other antigens that are not of interest that the antibody is useful for, at minimum, detecting the presence of the antigen of interest in a particular type of biological sample. In certain methods employing the antibody, such as therapeutic applications, a higher degree of specificity in binding may be desirable. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to discriminate effectively between the desired antigens and cross-reacting polypeptides. One characteristic that influences the specificity of an antibody:antigen interaction is the affinity of the antibody for the antigen.
  • antibodies will have an affinity (a dissociation constant) of about 10 "6 , 10 "7 , 10 "8 , 10 " 9 M or less.
  • the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding.
  • a variety of different techniques are available for testing interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the BiacoreTM binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), western blots, immunoprecipitation assays, and immunohi stochemi stry .
  • a nucleic acid compound may be single or double stranded.
  • a double stranded compound may also include regions of overhang or non-complementarity, where one or the other of the strands is single stranded.
  • a single stranded compound may include regions of self-complementarity, meaning that the compound forms a so-called "hairpin” or "stem-loop” structure, with a region of double helical structure.
  • a nucleic acid compound may comprise a nucleotide sequence that is complementary to a region consisting of no more than 1000, no more than 500, no more than 250, no more than 100, or no more than 50, 35, 25, 22, 20, 18 or 15 nucleotides of the full-length ActRII nucleic acid sequence or activin P A , P B , P C , or P E nucleic acid sequence.
  • the region of complementarity will preferably be at least 8 nucleotides, and optionally about 18 to 35 nucleotides.
  • a region of complementarity may fall within an intron, a coding sequence or a noncoding sequence of the target transcript, such as the coding sequence portion.
  • a nucleic acid compound will have a length of about 8 to about 500 nucleotides or base pairs in length, and optionally the length will be about 14 to about 50 nucleotides.
  • a nucleic acid may be a DNA (particularly for use as an antisense), RNA or RNA:DNA hybrid. Any one strand may include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA.
  • a double stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any one strand may also include a mixture of DNA and RNA, as well as modified forms that cannot readily be classified as either DNA or RNA.
  • a nucleic acid compound may include any of a variety of modifications, including one or modifications to the backbone (the sugar-phosphate portion in a natural nucleic acid, including internucleotide linkages) or the base portion (the purine or pyrimidine portion of a natural nucleic acid).
  • An antisense nucleic acid compound will preferably have a length of about 15 to about 30 nucleotides and will often contain one or more modifications to improve characteristics such as stability in the serum, in a cell or in a place where the compound is likely to be delivered, such as the stomach in the case of orally delivered compounds and the lung for inhaled compounds.
  • the strand complementary to the target transcript will generally be RNA or modifications thereof.
  • the other strand may be RNA, DNA or any other variation.
  • the duplex portion of double stranded or single stranded "hairpin" RNAi construct will generally have a length of 18 to 40 nucleotides in length and optionally about 21 to 23 nucleotides in length, so long as it serves as a Dicer substrate.
  • Catalytic or enzymatic nucleic acids may be ribozymes or DNA enzymes and may also contain modified forms.
  • Nucleic acid compounds may inhibit expression of the target by about 50%, 75%, 90% or more when contacted with cells under physiological conditions and at a concentration where a nonsense or sense control has little or no effect. Preferred concentrations for testing the effect of nucleic acid compounds are 1 , 5 and 10 micromolar. Nucleic acid compounds may also be tested for effects on, for example, red blood cell levels.
  • the present invention relates to the use of ActRII polypeptides (e.g., soluble ActRIIa or ActRIIb polypeptides) and activin polypeptides to identify compounds (agents) which are agonist or antagonists of the activin- ActRIIa and/or activin ActRIIb signaling pathway.
  • ActRII polypeptides e.g., soluble ActRIIa or ActRIIb polypeptides
  • activin polypeptides e.g., soluble ActRIIa or ActRIIb polypeptides
  • activin polypeptides e.g., soluble ActRIIa or ActRIIb polypeptides
  • Compounds identified through this screening can be tested to assess their ability to modulate red blood cell, hemoglobin and/or reticulocyte levels in vivo or in vitro. These compounds can be tested, for example, in animal models.
  • high-throughput screening of compounds can be carried out to identify agents that perturb activin or ActRII-mediated effects on a selected cell line.
  • the assay is carried out to screen and identify compounds that specifically inhibit or reduce binding of an ActRIIa or ActRIIb polypeptide to activin.
  • the assay can be used to identify compounds that enhance binding of an ActRIIa or ActRIIb polypeptide to activin.
  • the compounds can be identified by their ability to interact with an activin, ActRIIb polypeptide, or ActRIIa polypeptide.
  • test compounds (agents) of the invention may be created by any combinatorial chemical method.
  • the subject compounds may be naturally occurring biomolecules synthesized in vivo or in vitro.
  • Compounds (agents) to be tested for their ability to act as modulators of tissue growth can be produced, for example, by bacteria, yeast, plants or other organisms (e.g., natural products), produced chemically (e.g., small molecules, including peptidomimetics), or produced recombinantly.
  • Test compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleic acid molecules.
  • the test agent is a small organic molecule having a molecular weight of less than about 2,000 Daltons.
  • test compounds of the invention can be provided as single, discrete entities, or provided in libraries of greater complexity, such as made by combinatorial chemistry.
  • libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds.
  • Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps.
  • the compounds may be optionally derivatized with other compounds and have derivatizing groups that facilitate isolation of the compounds.
  • Non- limiting examples of derivatizing groups include biotin, fluorescein, digoxygenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S transferase (GST), photoactivatible crosslinkers or any combinations thereof.
  • the effects of cellular toxicity or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity between an ActRIIa polypeptide and activin and/or between an ActRIIb polypeptide and activin.
  • the compound of interest is contacted with an isolated and purified ActRIIa polypeptide which is ordinarily capable of binding to activin.
  • an isolated and purified ActRIIa polypeptide which is ordinarily capable of binding to activin.
  • a composition containing an ActRIIa ligand is then added to the mixture of the compound and ActRIIa polypeptide.
  • Detection and quantification of ActRIIa/activin complexes provides a means for determining the compound's efficacy at inhibiting (or potentiating) complex formation between the ActRIIa polypeptide and activin.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay can also be performed to provide a baseline for comparison.
  • isolated and purified activin is added to a composition containing the ActRIIa polypeptide, and the formation of ActRIIa/activin complex is quantitated in the absence of the test compound.
  • the order in which the reactants may be admixed can be varied, and can be admixed simultaneously.
  • cellular extracts and lysates may be used to render a suitable cell- free assay system.
  • Compounds that affect ActRIIb signaling may be identified in a similar manner using an ActRIIb polypeptide and an ActRIIb ligand.
  • Complex formation between the ActRII polypeptide and activin may be detected by a variety of techniques. For instance, modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled (e.g., 32 P, 35 S, 14 C or 3 H), fluorescently labeled (e.g., FITC), or enzymatically labeled ActRIIa or ActRIIb polypeptide or activin, by immunoassay, or by chromatographic detection.
  • detectably labeled proteins such as radiolabeled (e.g., 32 P, 35 S, 14 C or 3 H), fluorescently labeled (e.g., FITC), or enzymatically labeled ActRIIa or ActRIIb polypeptide or activin, by immunoassay, or by chromatographic detection.
  • the present invention contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays in measuring, either directly or indirectly, the degree of interaction between an ActRII polypeptide and its binding protein.
  • FRET fluorescence resonance energy transfer
  • other modes of detection such as those based on optical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No. 5,677,196), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with many embodiments of the invention.
  • the present invention contemplates the use of an interaction trap assay, also known as the "two hybrid assay," for identifying agents that disrupt or potentiate interaction between an ActRIl polypeptide and its binding protein.
  • the present invention contemplates the use of reverse two hybrid systems to identify compounds (e.g., small molecules or peptides) that dissociate interactions between an ActRIl polypeptide and its binding protein.
  • the subject compounds are identified by their ability to interact with an ActRIl or activin polypeptide of the invention.
  • the interaction between the compound and the ActRIIa, ActRIIb, or activin polypeptide may be covalent or non-covalent.
  • such interaction can be identified at the protein level using in vitro biochemical methods, including photo-crosslinking, radiolabeled ligand binding, and affinity chromatography (Jakoby WB et al., 1974, Methods in Enzymology 46: 1).
  • the compounds may be screened in a mechanism based assay, such as an assay to detect compounds which bind to an activin or ActRIl polypeptide. This may include a solid phase or fluid phase binding event.
  • the gene encoding an activin or ActRIl polypeptide can be transfected with a reporter system (e.g., ⁇ -galactosidase, luciferase, or green fluorescent protein) into a cell and screened against the library optionally by a high throughput screening or with individual members of the library.
  • a reporter system e.g., ⁇ -galactosidase, luciferase, or green fluorescent protein
  • Other mechanism based binding assays may be used, for example, binding assays which detect changes in free energy. Binding assays can be performed with the target fixed to a well, bead or chip or captured by an immobilized antibody or resolved by capillary electrophoresis. The bound compounds may be detected usually using colorimetric or fluorescence or surface plasmon resonance.
  • activin-ActRII antagonists e.g., ActRIIa or ActRIIb polypeptides
  • the present invention provides methods of treating or preventing anemia in an individual in need thereof by administering to the individual a therapeutically effective amount of an activin-ActRIIa antagonist, such as an ActRIIa polypeptide, or a therapeutically effective amount of an activin-ActRIIb antagonist, such as an ActRIIb polypeptide.
  • the present invention provides methods of promoting red blood cell formation in an individual by administering to the individual a therapeutically effective amount of an activin-ActRII antagonist, particularly an ActRII polypeptide. These methods may be used for therapeutic and prophylactic treatments of mammals, and particularly humans.
  • a therapeutic that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • treating includes prophylaxis of the named condition or amelioration or elimination of the condition once it has been established. In either case, prevention or treatment may be discerned in the diagnosis provided by a physician or other health care provider and the intended result of administration of the therapeutic agent.
  • activin-ActRIIa antagonists and activin-ActRIIb antagonists may be used to increase red blood cell, hemoglobin or reticulocyte levels in healthy individuals, and such antagonists may be used in selected patient populations.
  • appropriate patient populations include those with undesirably low red blood cell or hemoglobin levels, such as patients having an anemia, and those that are at risk for developing undesirably low red blood cell or hemoglobin levels, such as those patients that are about to undergo major surgery or other procedures that may result in substantial blood loss.
  • a patient with adequate red blood cell levels is treated with an activin-ActRIIa antagonist to increase red blood cell levels, and then blood is drawn and stored for later use in transfusions.
  • a patient with adequate red blood cell levels is treated with an activin- ActRIIb antagonist to increase red blood cell levels, and then blood is drawn and stored for later use in transfusions.
  • Activin-ActRIl antagonists disclosed herein, and particularly ActRIIa-Fc and ActRIIb proteins may be used to increase red blood cell levels in patients having an anemia.
  • a level of less than normal for the appropriate age and gender category may be indicative of anemia, although individual variations are taken into account.
  • a hemoglobin level of 12 g/dl is generally considered the lower limit of normal in the general adult population.
  • Potential causes include blood-loss, nutritional deficits, medication reaction, various problems with the bone marrow and many diseases.
  • anemia has been associated with a variety of disorders that include, for example, chronic renal failure, myelodysplastic syndrome, rheumatoid arthritis, bone marrow transplantation.
  • Anemia may also be associated with the following conditions: solid tumors (e.g. breast cancer, lung cancer, colon cancer); tumors of the lymphatic system (e.g. chronic lymphocyte leukemia, non-Hodgkins and Hodgkins lymphomas); tumors of the hematopoietic system (eg. leukemia, myelodysplastic syndrome, multiple myeloma); radiation therapy; chemotherapy (e.g.
  • platinum containing regimens inflammatory and autoimmune diseases, including, but not limited to, rheumatoid arthritis, other inflammatory arthritides, systemic lupus erythematosis (SLE), acute or chronic skin diseases (e.g. psoriasis), inflammatory bowel disease (e.g. Crohn's disease and ulcerative colitis); acute or chronic renal disease or failure including idiopathic or congenital conditions; acute or chronic liver disease; acute or chronic bleeding; situations where transfusion of red blood cells is not possible due to patient allo- or auto-antibodies and/or for religious reasons (e.g. some
  • infections e.g. malaria, osteomyelitis
  • hemoglobinopathies including, for example, sickle cell disease, thalassemias
  • drug use or abuse e.g. alcohol misuse
  • pediatric patients with anemia from any cause to avoid transfusion and elderly patients or patients with underlying cardiopulmonary disease with anemia who cannot receive transfusions due to concerns about circulatory overload.
  • Patients may be treated with a dosing regimen intended to restore the patient to a target hemoglobin level, usually between about 10 g/dl and about 12.5 g/dl, and typically about 1 1.0 g/dl (see also Jacobs et al. (2000) Nephrol Dial Transplant 15, 15-19), although lower target levels may cause fewer cardiovascular side effects.
  • hematocrit levels percentage of the volume of a blood sample occupied by the cells
  • Hematocrit levels for healthy individuals range from 41 to 51% for adult males and from 35 to 45% for adult females.
  • Target hematocrit levels are usually around 30-33%.
  • hemoglobin/hematocrit levels vary from person to person. Thus, optimally, the target hemoglobin/hematocrit level can be individualized for each patient.
  • an activin-ActRIIa antagonist may be useful for increasing red blood cell and hemoglobin levels in patients that do not respond well to Epo.
  • an activin-ActRIIa antagonist may be beneficial for a patient in which administering of a normal to increased (>300 IU/kg/week) dose of Epo does not result in the increase of hemoglobin level up to the target level.
  • Patients with an inadequate Epo response are found for all types of anemia, but higher numbers of non- responders have been observed particularly frequently in patients with cancers and patients with end-stage renal disease.
  • An inadequate response to Epo can be either constitutive (i.e.
  • the activin-ActRII antagonists may also be used to treat patients that are susceptible to adverse effects of Epo.
  • the primary adverse effects of Epo are an excessive increase in the hematocrit or hemoglobin levels and polycythemia. Elevated hematocrit levels can lead to hypertension (more particularly aggravation of hypertension) and vascular thrombosis.
  • activin-ActRII antagonists e.g., ActRIIa and ActRIlb polypeptides
  • a pharmaceutically acceptable carrier e.g., an ActRII polypeptide can be administered alone or as a component of a pharmaceutical formulation (therapeutic composition).
  • the subject compounds may be formulated for administration in any convenient way for use in human or veterinary medicine.
  • the therapeutic method of the invention includes administering the composition systemically, or locally as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen- free, physiologically acceptable form.
  • Therapeutically useful agents other than the activin- ActRII antagonists which may also optionally be included in the composition as described above, may be administered simultaneously or sequentially with the subject compounds (e.g., ActRIIa and ActRIIb polypeptides) in the methods of the invention.
  • activin-ActRII antagonists will be administered parenterally.
  • compositions suitable for parenteral administration may comprise one or more ActRII polypeptides in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions of the present invention may include a matrix capable of delivering one or more therapeutic compounds (e.g., ActRIIa or ActRIIb polypeptides) to a target tissue site (e.g., bone marrow), providing a structure for the developing tissue and optimally capable of being resorbed into the body.
  • the matrix may provide slow release of the ActRII polypeptides.
  • Such matrices may be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.
  • Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid and polyanhydrides.
  • Other potential materials are biodegradable and biologically well defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are non-biodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.
  • Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate.
  • the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
  • methods of the invention can be administered for orally, e.g., in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an agent as an active ingredient.
  • An agent may also be administered as a bolus, electuary or paste.
  • one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents,
  • pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: (1 ) fillers or
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents,
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • compositions of the invention may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
  • the dosage regimen will be determined by the attending physician considering various factors which modify the action of the subject compounds of the invention (e.g., ActRIIa and ActRIlb polypeptides).
  • the various factors include, but are not limited to, the patient's red blood cell count, hemoglobin level or other diagnostic assessments, the desired target red blood cell count, the patient's age, sex, and diet, the severity of any disease that may be contributing to a depressed red blood cell level, time of administration, and other clinical factors.
  • the addition of other known growth factors to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of red blood cell and hemoglobin levels, as well as assessments of reticulocyte levels and other indicators of the hematopoietic process.
  • a dosing scheme may be designed to deliver a serum concentration of between about 100 and 1000 ng/ml over a period of about 20 to 30 days.
  • serum levels of 200 ng/ml may be achieved with a single dose of 0.1 mg/kg or greater and serum levels of 1000 ng/ml may be achieved with a single dose of 0.3 mg/kg or greater.
  • the observed serum half-life of the molecule is between about 20 and 30 days, substantially longer than most Fc fusion proteins, and thus a sustained effective serum level may be achieved, for example, by dosing with about 0.05 to 0.5 mg/kg on a weekly or biweekly basis, or higher doses may be used with longer intervals between dosings. For example, doses of 0.1 to 1 mg/kg might be used on a monthly or bimonthly basis.
  • the present invention also provides gene therapy for the in vivo production of ActRIl polypeptides.
  • Such therapy would achieve its therapeutic effect by introduction of the ActRIIa or ActRIlb polynucleotide sequences into cells or tissues having the disorders as listed above.
  • Delivery of ActRIl polynucleotide sequences can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system.
  • Preferred for therapeutic delivery of ActRII polynucleotide sequences is the use of targeted liposomes.
  • RNA virus such as a retrovirus
  • the retroviral vector may be a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • Retroviral vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Retroviral vectors can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody.
  • specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing the ActRII polynucleotide.
  • tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • Another targeted delivery system for ActRII polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (see e.g., Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Methods for efficient gene transfer using a liposome vehicle, are known in the art, see e.g., Mannino, et al., Biotechniques, 6:682, 1988.
  • the composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
  • the targeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art.
  • Applicants constructed a soluble ActRIIa fusion protein that has the extracellular domain of human ActRIIa fused to a human or mouse Fc domain with a minimal linker in between.
  • the constructs are referred to as ActRIIa-hFc and ActRIIa-mFc, respectively.
  • ActRIIa-hFc is shown below as purified from CHO cell lines (SEQ ID NO: 7): ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCF ATWKNISGSIEIVKQG CWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPK PPTGGGTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP VPIEKTISKAKGOPREPQVYTLPPSREEMTKNOVSLTCLVKGFYPSDIAVEWESNGOP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSLSL SPGK
  • the ActRIIa-hFc and ActRIIa-mFc proteins were expressed in CHO cell lines. Three different leader sequences were considered:
  • the selected form employs the TPA leader and has the following unprocessed amino acid sequence:
  • This polypeptide is encoded by the following nucleic acid sequence:
  • ActRIIa-hFc and ActRIIa-mFc showed a high affinity for ligands, particularly activin A.
  • GDF-1 1 or Activin A (“ActA") were immobilized on a Biacore CM5 chip using standard amine coupling procedure.
  • ActA Activin A
  • ActA Activin A
  • ActIa-hFc and ActRIIa-mFc proteins were loaded onto the system, and binding was measured.
  • ActRIIa-hFc bound to activin with a dissociation constant (K 0 ) of 5x10 12 , and the protein bound to GDFl 1 with a K D of 9.96x10 9 . See figure 2.
  • ActRIIa-mFc behaved similarly.
  • the ActRIIa-hFc was very stable in pharmacokinetic studies.
  • Rats were dosed with 1 mg/kg, 3 mg/kg or 10 mg/kg of ActRIIa-hFc protein and plasma levels of the protein were measured at 24, 48, 72, 144 and 168 hours. In a separate study, rats were dosed at 1 mg/kg, 10 mg/kg or 30 mg/kg.
  • ActRIIa-hFc had an 11-14 day serum half life and circulating levels of the drug were quite high after two weeks (1 1 ⁇ g/ml, 1 10 ⁇ g/ml or 304 ⁇ g/ml for initial administrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively.)
  • the plasma half life was substantially greater than 14 days and circulating levels of the drug were 25 ⁇ g/ml, 304 ⁇ g/ml or 1440 ⁇ g/ml for initial administrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively.
  • Example 2 ActRIIa-hFc Increases Red Blood Cell Levels in Non-Human Primates
  • the study employed four groups of five male and five female cynomolgus monkeys each, with three per sex per group scheduled for termination on Day 29, and two per sex per group scheduled for termination on Day 57.
  • Each animal was administered the vehicle (Group I) or ActRIla-Fc at doses of 1 , 10, or 30 mg/kg (Groups 2, 3 and 4, respectively) via intravenous (IV) injection on Days 1 , 8, 15 and 22.
  • the dose volume was maintained at 3 mL/kg.
  • Various measures of red blood cell levels were assessed two days prior to the first administration and at days 15, 29 and 57 (for the remaining two animals) after the first administration.
  • the ActRIIa-hFc causes statistically significant increases in mean red blood cell parameters (red blood cell count [RBC], hemoglobin [HGB], and hematocrit [HCT]) for males and females, at all dose levels and time points throughout the study, with accompanying elevations in absolute and relative reticulocyte counts (ARTC; RTC). See Figures 3 - 6. Statistical significance was calculated for each treatment group relative to the mean for the treatment group at baseline.
  • RBC red blood cell count
  • HGB hemoglobin
  • HCT hematocrit
  • the ActRIIa-hFc fusion protein described in Example 1 was administered to human patients in a randomized, double-blind, placebo-controlled study that was conducted to evaluate, primarily, the safety of the protein in healthy, postmenopausal women. Forty-eight subjects were randomized in cohorts of 6 to receive either a single dose of ActRIIa-hFc or placebo (5 active: 1 placebo). Dose levels ranged from 0.01 to 3.0 mg/kg intravenously (IV) and 0.03 to 0.1 mg/kg subcutaneously (SC). All subjects were followed for 120 days. In addition to pharmacokinetic (PK) analyses, the biologic activity of ActRIIa-hFc was also assessed by measurement of biochemical markers of bone formation and resorption, and FSH levels.
  • PK pharmacokinetic
  • hemoglobin and RBC numbers were examined in detail for all subjects over the course of the study and compared to the baseline levels. Platelet counts were compared over the same time as the control. There were no clinically significant changes from the baseline values over time for the platelet counts.
  • PK analysis of ActRIIa-hFc displayed a linear profile with dose, and a mean half-life of approximately 25-32 days.
  • the area-under-curve (AUC) for ActRIIa-hFc was linearly related to dose, and the absorption after SC dosing was essentially complete (see Figures 7 and 8).
  • ActRIIa-hFc caused a rapid, sustained dose-dependent increase in serum levels of bone-specific alkaline phosphatase (BAP), which is a marker for anabolic bone growth, and a dose- dependent decrease in C-terminal type 1 collagen telopeptide and tartrate-resistant acid phosphatase 5b levels, which are markers for bone resorption.
  • BAP bone-specific alkaline phosphatase
  • Other markers, such as PlNP showed inconclusive results.
  • BAP levels showed near saturating effects at the highest dosage of drug, indicating that half-maximal effects on this anabolic bone biomarker could be achieved at a dosage of 0.3 mg/kg, with increases ranging up to 3 mg/kg.
  • Example 4 Alternative ActRIIa-Fc Proteins
  • ActRIIa variants that may be used according to the methods described herein are described in the International Patent Application published as WO2006/012627 (see e.g., pp. 55-58), incorporated herein by reference in its entirety.
  • An alternative construct may have a deletion of the C-terminal tail (the final 15 amino acids of the extracellular domain of ActRIIa. The sequence for such a construct is presented below (Fc portion underlined)(SEQ ID NO: 12):
  • a co-crystal structure of Activin and extracellular ActRIIb did not show any role for the final (C-terminal) 15 amino acids (referred to as the "tail” herein) of the extracellular domain in ligand binding.
  • This sequence failed to resolve on the crystal structure, suggesting that these residues are present in a flexible loop that did not pack uniformly in the crystal. Thompson et al. EMBO J. 2003 Apr 1 ;22(7):1555-66. This sequence is also poorly conserved between ActRIIb and ActRIIa.
  • the background ActRIIb-Fc fusion has the sequence (Fc portion underlined)(SEQ ID NO:20): SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVK KGCWLDDFNCYDROECVATEENPOVYFCCCEGNFCNERFTHLPEAGGGTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPVPIEKTISKAKGOP REPOVYTLPPSREEMTKNOVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOKSLSLSPGK
  • ActRlIb-Fc has a sequence (Fc portion underlined)(SEQ ID N0:21): SGRGEAETRECIYYN ANWELERTNQSGLERCEGEQDKRLHCYAS WANSSGTIELVK KGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPP TAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKA LPVPIEKTISKAKGOPREPOVYTLPPSREEMTKNOVSLTCLVKGFYPSDIAVEWESNG OPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWOOGNVFSCSVMHEALHNHYTOK
  • ActRIIb a variants that may be used according to the methods described herein are described in the International Patent Application published as WO2006/012627 (see e.g., pp. 59-60), incorporated herein by reference in its entirety.
  • ActRIIb-hFc (IgGl) was administered once a week for 1 -month to male and female cynomolgus monkeys by subcutaneous injection. Forty-eight cynomolgus monkeys (24/sex) were assigned to one of four treatment groups (6 animals/sex/group) and were administered subcutaneous injections of either vehicle or ActRIIb-hFc at 3, 10, or 30 mg/kg once weekly for 4 weeks (total of 5 doses). Parameters evaluated included general clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis). ActRIIb-hFc caused statistically significant elevated mean absolute reticulocyte values by day 15 in treated animals.
  • ActRIIb-hFc caused several hematological changes, including elevated mean absolute reticulocyte and red blood cell distribution width values and lower mean corpuscular hemoglobin concentration. All treated groups and both sexes were affected. These effects are consistent with a positive effect of ActRIIb-hFc on the release of immature reticulocytes from the bone marrow. This effect was reversed after drug was washed out of the treated animals (by study day 56). Accordingly, we conclude that ActRIIb-hFc stimulates erythropoiesis.

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Abstract

L'invention porte sur sous certains aspects, sur des compositions et méthodes accroissant les niveaux de globules rouges et/ou d'hémoglobine chez les vertébrés, dont les rongeurs les primates et en particulier l'homme.
EP07863068A 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour le traitement de l'anémie Active EP2124999B1 (fr)

Priority Applications (11)

Application Number Priority Date Filing Date Title
DK11195130.7T DK2468290T3 (en) 2006-12-18 2007-12-18 Activin-ActRII antagonists for use in the treatment of anemia
EP11195130.7A EP2468290B1 (fr) 2006-12-18 2007-12-18 Antagonistes de L'Activine-ActRII pour leur utilisation dans le traitement de l'anémie
PL11195130T PL2468290T3 (pl) 2006-12-18 2007-12-18 Antagoniści aktywiny-ActRII do stosowania w leczeniu niedokrwistości
PL07863068T PL2124999T3 (pl) 2006-12-18 2007-12-18 Antagoniści aktywiny-actrii i zastosowania do leczenia niedokrwistości
EP21205033.0A EP4026553A1 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour accroitre les niveaux de globules rouges
EP11195282A EP2446896A1 (fr) 2006-12-18 2007-12-18 Antagonistes de L'Activine-ActRII pour leur utilisation pour accroître les niveaux de globules rouges, pour accroître les niveaux de réticulocytes ou pour promouvoir l'érythropoièse
EP17182870.0A EP3320911B1 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour acrroitre les niveaux de globules rouges
EP11195140.6A EP2468291B1 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour acrroitre les niveaux de globules rouges
SI200731110T SI2124999T1 (sl) 2006-12-18 2007-12-18 Activin-actrii antagonisti in uporaba za zdravljenje anemije
MEP-2016-200A ME02335B (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour le traitement de l'anémie
CY20121101136T CY1114827T1 (el) 2006-12-18 2012-11-26 Ανταγωνιστες ακτιβινης-actrii και η χρηση τους στην αντιμετωπιση της αναιμιας

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US87568206P 2006-12-18 2006-12-18
PCT/US2007/025868 WO2008076437A2 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour accroître les niveaux de globules rouges

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EP21205033.0A Division EP4026553A1 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour accroitre les niveaux de globules rouges
EP11195140.6A Division EP2468291B1 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour acrroitre les niveaux de globules rouges
EP11195140.6A Division-Into EP2468291B1 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour acrroitre les niveaux de globules rouges
EP11195282A Division-Into EP2446896A1 (fr) 2006-12-18 2007-12-18 Antagonistes de L'Activine-ActRII pour leur utilisation pour accroître les niveaux de globules rouges, pour accroître les niveaux de réticulocytes ou pour promouvoir l'érythropoièse
EP17182870.0A Division EP3320911B1 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour acrroitre les niveaux de globules rouges
EP11195130.7A Division-Into EP2468290B1 (fr) 2006-12-18 2007-12-18 Antagonistes de L'Activine-ActRII pour leur utilisation dans le traitement de l'anémie
EP11195130.7A Division EP2468290B1 (fr) 2006-12-18 2007-12-18 Antagonistes de L'Activine-ActRII pour leur utilisation dans le traitement de l'anémie

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EP2124999A2 true EP2124999A2 (fr) 2009-12-02
EP2124999B1 EP2124999B1 (fr) 2012-10-03

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EP21205033.0A Withdrawn EP4026553A1 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour accroitre les niveaux de globules rouges
EP07863068A Active EP2124999B1 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour le traitement de l'anémie
EP11195140.6A Active EP2468291B1 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour acrroitre les niveaux de globules rouges
EP17182870.0A Active EP3320911B1 (fr) 2006-12-18 2007-12-18 Antagonistes de l'activine-actrii et ses utilisations pour acrroitre les niveaux de globules rouges
EP11195282A Withdrawn EP2446896A1 (fr) 2006-12-18 2007-12-18 Antagonistes de L'Activine-ActRII pour leur utilisation pour accroître les niveaux de globules rouges, pour accroître les niveaux de réticulocytes ou pour promouvoir l'érythropoièse

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Families Citing this family (82)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6989276B2 (en) 2001-05-10 2006-01-24 Battelle Energy Alliance, Llc Rapid classification of biological components
USRE46351E1 (en) 2001-05-10 2017-03-28 Battelle Energy Alliance, Llc Antibody profiling sensitivity through increased reporter antibody layering
ES2710048T3 (es) * 2004-07-23 2019-04-22 Acceleron Pharma Inc Anticuerpos antagonistas del receptor ActRII
BRPI0618947B1 (pt) 2005-11-23 2022-07-19 Acceleron Pharma Inc Polipeptídeo actriia que se liga à ativina, polinucleotídeo isolado, polinucleotídeo recombinante, métodopara preparar um polipeptídeo actriia que se liga à ativina, preparação farmacêutica e uso de uma proteína de fusão actriia-fc para promover o crescimento ósseo e inibir a ressorção óssea
US8128933B2 (en) 2005-11-23 2012-03-06 Acceleron Pharma, Inc. Method of promoting bone growth by an anti-activin B antibody
US20100028332A1 (en) * 2006-12-18 2010-02-04 Acceleron Pharma Inc. Antagonists of actriib and uses for increasing red blood cell levels
JP5415279B2 (ja) * 2006-12-18 2014-02-12 アクセルロン ファーマ， インコーポレイテッド アクチビン−ＡｃｔＲＩＩアンタゴニストおよび赤血球レベルを増加させるためのその使用
US8895016B2 (en) 2006-12-18 2014-11-25 Acceleron Pharma, Inc. Antagonists of activin-actriia and uses for increasing red blood cell levels
AU2008211007B2 (en) * 2007-02-01 2013-09-19 Acceleron Pharma Inc. Activin-ActRIIa antagonists and uses for treating or preventing breast cancer
TW201803890A (zh) * 2007-02-02 2018-02-01 艾瑟勒朗法瑪公司 衍生自ＡｃｔＲＩＩＢ的變體與其用途
ES2756725T3 (es) 2007-02-09 2020-04-27 Acceleron Pharma Inc Composiciones farmacéuticas que comprenden antagonistas de Activina-ActRIIA
US7947646B2 (en) 2007-03-06 2011-05-24 Amgen Inc. Variant activin receptor polypeptides
US20080286881A1 (en) * 2007-05-14 2008-11-20 Apel William A Compositions and methods for combining report antibodies
CA2699936A1 (fr) 2007-09-18 2009-03-26 Acceleron Pharma Inc. Antagonistes de l'activine-actriia et utilisations pour reduire ou empecher la secretion de fsh
EP3363453A1 (fr) * 2008-06-26 2018-08-22 Acceleron Pharma Inc. Actriia soluble en tant qu'antagoniste activine-actriia pour utilisation dans le traitement de l'anémie ou de maladies osseuses
CA3049354A1 (fr) * 2008-06-26 2009-12-30 Acceleron Pharma Inc. Antagonistes d'actriib et utilisations pour augmenter les taux d'erythrocytes
AU2015202035B2 (en) * 2008-08-14 2017-03-23 Acceleron Pharma Inc. Use of GDF traps to increase red blood cell levels
US8216997B2 (en) 2008-08-14 2012-07-10 Acceleron Pharma, Inc. Methods for increasing red blood cell levels and treating anemia using a combination of GDF traps and erythropoietin receptor activators
PL3750552T3 (pl) * 2008-08-14 2023-11-06 Acceleron Pharma Inc. Pułapki gdf
US20110262466A1 (en) * 2008-10-16 2011-10-27 The Trustees Of The University Of Pennsylvania Compositions containing thrombomodulin domains and uses thereof
LT2370463T (lt) 2008-11-26 2016-12-12 Amgen Inc. Stabilizuotas aktivino iib receptoriaus variantas
AU2010204985A1 (en) 2009-01-13 2011-08-04 Acceleron Pharma Inc. Methods for increasing adiponectin
BRPI1010587A2 (pt) 2009-06-08 2019-04-09 Acceleron Pharma Inc. métodos para aumentar adipócitos termogênicos
KR20190090049A (ko) 2009-06-12 2019-07-31 악셀레론 파마 인코포레이티드 절두된 ＡｃｔＲＩＩＢ-ＦＣ 융합 단백질
IL287990B (en) * 2009-08-13 2022-07-01 Acceleron Pharma Inc Combined use of gdf traps and erythropoietin receptor activators to increase red blood cell levels
EP2475427B1 (fr) * 2009-09-09 2016-11-30 Acceleron Pharma, Inc. Antagonistes d'actriib, et dosage et administration associés
US8969009B2 (en) * 2009-09-17 2015-03-03 Vicki S. Thompson Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual
US9410965B2 (en) * 2009-09-17 2016-08-09 Battelle Energy Alliance, Llc Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual
US20110129469A1 (en) * 2009-11-03 2011-06-02 Acceleron Pharma Inc. Methods for treating fatty liver disease
WO2011063018A1 (fr) 2009-11-17 2011-05-26 Acceleron Pharma Inc. Protéines actriib et variants et utilisations de celles-ci se rapportant à l'induction de l'utrophine pour une thérapie de la dystrophie musculaire
WO2012019168A2 (fr) 2010-08-06 2012-02-09 Moderna Therapeutics, Inc. Acides nucléiques modifiés et leurs procédés d'utilisation
WO2012027065A2 (fr) * 2010-08-27 2012-03-01 Celgene Corporation Polythérapie pour traiter des maladies
DK3590949T3 (da) 2010-10-01 2022-07-11 Modernatx Inc Ribonukleinsyrer indeholdende n1-methyl-pseudouracil og anvendelse heraf
AU2011326586A1 (en) 2010-11-08 2013-05-30 Acceleron Pharma, Inc. ActRIIA binding agents and uses thereof
US8710200B2 (en) 2011-03-31 2014-04-29 Moderna Therapeutics, Inc. Engineered nucleic acids encoding a modified erythropoietin and their expression
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
RU2648950C2 (ru) 2011-10-03 2018-04-02 Модерна Терапьютикс, Инк. Модифицированные нуклеозиды, нуклеотиды и нуклеиновые кислоты и их применение
US20130156849A1 (en) 2011-12-16 2013-06-20 modeRNA Therapeutics Modified nucleoside, nucleotide, and nucleic acid compositions
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
WO2013151667A1 (fr) 2012-04-02 2013-10-10 modeRNA Therapeutics Polynucléotides modifiés
US9303079B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9809636B2 (en) 2012-04-06 2017-11-07 Acceleron Pharma Inc. Methods for increasing red blood cell levels comprising administering BMP9
GB201206559D0 (en) 2012-04-13 2012-05-30 Ucl Business Plc Polypeptide
JP6214537B2 (ja) * 2012-08-21 2017-10-25 国立大学法人九州大学 貧血患者の貧血の要因を検出するためのバイオマーカー
NZ747350A (en) * 2012-10-24 2020-07-31 Celgene Corp Methods for treating anemia
IL267663B (en) * 2012-10-24 2022-09-01 Celgene Corp A biomarker for use in the treatment of anemia
BR112015009948B1 (pt) * 2012-11-02 2022-12-06 Celgene Corporation Uso de um inibidor de actrii
RS63237B1 (sr) 2012-11-26 2022-06-30 Modernatx Inc Terminalno modifikovana rnk
MX2015009901A (es) 2013-02-01 2016-04-06 Santa Maria Biotherapeutics Inc Administración de un compuesto de antiactivina a a un sujeto.
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
KR20160067219A (ko) 2013-10-03 2016-06-13 모더나 세라퓨틱스, 인코포레이티드 저밀도 지단백질 수용체를 암호화하는 폴리뉴클레오타이드
MA39722A (fr) * 2014-03-21 2021-06-02 Acceleron Pharma Inc Composition pour son utilisation dans une methode de traitement ou de prevention de l'anemie par l'inhibition de l'activine b et du gdf11
US10010498B2 (en) 2014-06-04 2018-07-03 Acceleron Pharma Inc. Methods for treatment of amyotrophic lateral sclerosis with follistatin fusion proteins
KR102077286B1 (ko) 2014-06-04 2020-02-13 악셀레론 파마 인코포레이티드 폴리스타틴 폴리펩티드를 이용한 장애의 치료방법 및 치료를 위한 조성물
TN2016000553A1 (en) * 2014-06-13 2018-04-04 Acceleron Pharma Inc Methods and compositions for treating ulcers
MA41052A (fr) 2014-10-09 2017-08-15 Celgene Corp Traitement d'une maladie cardiovasculaire à l'aide de pièges de ligands d'actrii
EP3922259A1 (fr) 2014-10-30 2021-12-15 Acceleron Pharma Inc. Méthodes et compositions utilisant des polypeptides gdf15 pour augmenter le nombre de globules rouges sanguins
JP2018501307A (ja) * 2014-12-03 2018-01-18 セルジーン コーポレイション アクチビン−ＡｃｔＲＩＩアンタゴニスト及び貧血を治療するための使用
MA41119A (fr) * 2014-12-03 2017-10-10 Acceleron Pharma Inc Méthodes de traitement de syndromes myélodysplasiques et d'anémie sidéroblastique
WO2016123454A1 (fr) * 2015-01-29 2016-08-04 Board Of Trustees Of Miching State University Polypeptides cryptiques et leurs utilisations
US20180031579A1 (en) 2015-02-12 2018-02-01 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for predicting the responsiveness of a patient affected with malignant hematological disease to chemotherapy treatment and methods of treatment of such disease
CA2980757A1 (fr) 2015-03-26 2016-09-29 Acceleron Pharma Inc. Proteines de fusion associees a la follistatine et leurs utilisations
AU2016246708B2 (en) * 2015-04-06 2020-12-24 Acceleron Pharma Inc. ALK7:actRIIB heteromultimers and uses thereof
AU2016251640B2 (en) 2015-04-22 2020-07-16 Alivegen Inc. Novel hybrid ActRIIB ligand trap proteins for treating muscle wasting diseases
TWI762444B (zh) * 2015-05-13 2022-05-01 美商西建公司 使用ＡＣＴＲＩＩ配位體捕捉以治療β-地中海型貧血
JP2018522579A (ja) 2015-05-20 2018-08-16 セルジーン コーポレイション アクチビンＩＩ型受容体リガンドトラップを使用するβ−サラセミアのインビトロ細胞培養法
JP7188883B2 (ja) * 2015-07-06 2022-12-13 三菱瓦斯化学株式会社 樹脂組成物、プリプレグ、レジンシート、積層板、及びプリント配線板
WO2017079591A2 (fr) 2015-11-04 2017-05-11 Acceleron Pharma Inc. Méthodes pour augmenter les taux d'érythrocytes et traiter l'érythropoïèse inefficace
KR20180096645A (ko) * 2015-11-23 2018-08-29 악셀레론 파마 인코포레이티드 눈 질환의 치료 방법
WO2017177013A1 (fr) * 2016-04-06 2017-10-12 Acceleron Pharma Inc. Antagonistes d'alk7 et leurs utilisations
LT3496739T (lt) 2016-07-15 2021-05-25 Acceleron Pharma Inc. Kompozicijos ir būdai, skirti plaučių hipertenzijai gydyti
JP6944768B2 (ja) 2016-08-29 2021-10-06 エア・ウォーター株式会社 ペリクルの製造方法
AU2017338916B2 (en) 2016-10-05 2022-03-24 Acceleron Pharma Inc. Variant ActRIIB proteins and uses thereof
AU2017357944A1 (en) 2016-11-10 2019-06-20 Keros Therapeutics, Inc. Activin receptor type IIa variants and methods of use thereof
US10982000B2 (en) 2017-03-24 2021-04-20 Novartis Ag Methods for treating and/or reducing the likelihood of heart failure by administering anti-activin receptor type II (anti-ActRII) antibody
CN111801112A (zh) 2017-11-09 2020-10-20 科乐斯疗法公司 激活素受体iia型变体及其使用方法
CN112292144A (zh) 2018-01-12 2021-01-29 科乐斯疗法公司 激活素受体iib型变体及其使用方法
JP7405772B2 (ja) * 2018-05-09 2023-12-26 ケロス セラピューティクス インコーポレイテッド アクチビンｉｉａ型受容体変異体および同変異体を含む医薬組成物
EP4065607A1 (fr) 2019-11-29 2022-10-05 Kymab Limited Traitement de surcharge de fer physiologique
CA3226249A1 (fr) * 2021-08-16 2023-02-23 Thermolife International, Llc Compositions de supplement de fer et leurs procedes d'utilisation
US11945856B2 (en) 2022-01-28 2024-04-02 35Pharma Inc. Activin receptor type IIB variants and uses thereof

Family Cites Families (81)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US71927A (en) * 1867-12-10 Improvement in apparatus fob turning on gas
JPH0637520B2 (ja) 1985-07-03 1994-05-18 味の素株式会社 ポリペプチド
US4973577A (en) 1986-04-04 1990-11-27 The Salk Institute For Biological Studies FSH-releasing peptides
US5080891A (en) 1987-08-03 1992-01-14 Ddi Pharmaceuticals, Inc. Conjugates of superoxide dismutase coupled to high molecular weight polyalkylene glycols
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US5198346A (en) 1989-01-06 1993-03-30 Protein Engineering Corp. Generation and selection of novel DNA-binding proteins and polypeptides
US5096815A (en) 1989-01-06 1992-03-17 Protein Engineering Corporation Generation and selection of novel dna-binding proteins and polypeptides
WO1992004913A1 (fr) * 1990-09-13 1992-04-02 Children's Hospital Medical Center Of Northern California Procede d'augmentation de la production de globules rouges par traitement a l'aide d'activine ou de peptides apparentes a l'activine
US5118667A (en) 1991-05-03 1992-06-02 Celtrix Pharmaceuticals, Inc. Bone growth factors and inhibitors of bone resorption for promoting bone formation
EP0542971A1 (fr) 1991-05-10 1993-05-26 The Salk Institute For Biological Studies CLONAGE ET PRODUCTION PAR RECOMBINAISON D'UN OU DE PLUSIEURS RECEPTEUR(S) DE LA SUPERFAMILLE ACTIVINE/FTC-$g(b)
US6162896A (en) 1991-05-10 2000-12-19 The Salk Institute For Biological Studies Recombinant vertebrate activin receptors
US5885794A (en) 1991-05-10 1999-03-23 The Salk Institute For Biological Studies Recombinant production of vertebrate activin receptor polypeptides and identification of receptor DNAs in the activin/TGF-β superfamily
US6692925B1 (en) 1992-11-17 2004-02-17 Ludwig Institute For Cancer Research Proteins having serine/threonine kinase domains, corresponding nucleic acid molecules, and their use
US5677196A (en) 1993-05-18 1997-10-14 University Of Utah Research Foundation Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays
US5831050A (en) 1993-06-07 1998-11-03 Creative Biomolecules, Inc. Morphogen cell surface receptor
CA2174098C (fr) 1993-10-14 2011-01-25 Douglas A. Melton Procede d'induction et de maintien de cellules neuronales
US5525490A (en) 1994-03-29 1996-06-11 Onyx Pharmaceuticals, Inc. Reverse two-hybrid method
US5658876A (en) 1994-04-28 1997-08-19 The General Hospital Corporation Activin antagonists as novel contraceptives
US5814565A (en) 1995-02-23 1998-09-29 University Of Utah Research Foundation Integrated optic waveguide immunosensor
DE69636866D1 (en) 1995-04-11 2007-03-15 Gen Hospital Corp Reverse "two-hybrid"-systeme
US6132988A (en) 1995-10-27 2000-10-17 Takeda Chemical Industries, Ltd. DNA encoding a neuronal cell-specific receptor protein
US6231880B1 (en) 1997-05-30 2001-05-15 Susan P. Perrine Compositions and administration of compositions for the treatment of blood disorders
AU8666398A (en) 1997-08-01 1999-02-22 Johns Hopkins University School Of Medicine, The Methods to identify growth differentiation factor (gdf) receptors
US6696260B1 (en) 1997-08-01 2004-02-24 The Johns Hopkins University School Of Medicine Methods to identify growth differentiation factor (GDF) binding proteins
US6891082B2 (en) 1997-08-01 2005-05-10 The Johns Hopkins University School Of Medicine Transgenic non-human animals expressing a truncated activintype II receptor
US6656475B1 (en) 1997-08-01 2003-12-02 The Johns Hopkins University School Of Medicine Growth differentiation factor receptors, agonists and antagonists thereof, and methods of using same
US6953662B2 (en) 1997-08-29 2005-10-11 Human Genome Sciences, Inc. Follistatin-3
WO2000043781A2 (fr) 1999-01-21 2000-07-27 Metamorphix, Inc. Inhibiteurs de facteurs de differenciation de la croissance et leurs utilisations
EP1174149A1 (fr) 1999-04-19 2002-01-23 Kyowa Hakko Kogyo Co., Ltd. Inhibiteur de proliferation cellulaire pour tumeurs androgeno-independantes
US6468543B1 (en) 1999-05-03 2002-10-22 Zymogenetics, Inc. Methods for promoting growth of bone using ZVEGF4
KR100791797B1 (ko) * 1999-12-15 2008-01-04 리서치 디벨럽먼트 파운데이션 인히빈 수용체로서의 베타글리칸 및 이의 용도
JP4487376B2 (ja) 2000-03-31 2010-06-23 味の素株式会社 腎疾患治療剤
EP2141243A3 (fr) 2000-10-16 2010-01-27 Brystol-Myers Squibb Company Supports de protéine pour mimer des anticorps et d'autres protéines de liaison
WO2002043759A2 (fr) 2000-12-01 2002-06-06 Wyeth Methode et composition permettant de moduler la croissance osseuse
TWI329129B (en) 2001-02-08 2010-08-21 Wyeth Corp Modified and stabilized gdf propeptides and uses thereof
DE60236861D1 (de) 2001-04-26 2010-08-12 Amgen Mountain View Inc Kombinatorische bibliotheken von monomerdomänen
AU2001286171B2 (en) 2001-05-25 2008-01-10 Serono Genetics Institute S.A. Human CDNAs and proteins and uses thereof
AUPR638101A0 (en) 2001-07-13 2001-08-09 Bioa Pty Limited Composition and method for treatment of disease
US6855344B2 (en) 2001-07-17 2005-02-15 Integrated Chinese Medicine Holdings, Ltd. Compositions and methods for prostate and kidney health and disorders, an herbal preparation
US7320789B2 (en) 2001-09-26 2008-01-22 Wyeth Antibody inhibitors of GDF-8 and uses thereof
US6784154B2 (en) 2001-11-01 2004-08-31 University Of Utah Research Foundation Method of use of erythropoietin to treat ischemic acute renal failure
US20030144203A1 (en) 2001-12-19 2003-07-31 Voyager Pharmaceutical Corporation Methods for slowing senescence and treating and preventing diseases associated with senescence
CA2476887A1 (fr) 2002-02-21 2003-09-04 Wyeth Proteine contenant un domaine de follistatine
US20030219846A1 (en) * 2002-02-28 2003-11-27 Pfizer Inc. Assay for activity of the ActRIIB kinase
JP2004121008A (ja) * 2002-09-30 2004-04-22 Toray Ind Inc ネコアクチビンａおよびそのサブユニット並びにその製造法
AR047392A1 (es) 2002-10-22 2006-01-18 Wyeth Corp Neutralizacion de anticuerpos contra gdf 8 y su uso para tales fines
US20040223966A1 (en) 2002-10-25 2004-11-11 Wolfman Neil M. ActRIIB fusion polypeptides and uses therefor
US20040197828A1 (en) 2003-03-26 2004-10-07 Gaddy Dana P. Method for diagnosis and treatment of bone turnover
WO2005028517A2 (fr) 2003-05-09 2005-03-31 The General Hospital Corporation PROTEINES DE FUSION SOLUBLES DU RECEPTEUR TGF- ss DE TYPE III
UA85055C2 (ru) 2003-06-02 2008-12-25 Уайт Применение ингибиторов миостатика (gdf8) в сочетании с кортикостероидами для лечения нервно-мышечных заболеваний
WO2005009460A2 (fr) * 2003-07-25 2005-02-03 Medexis, S.A. Composition pharmaceutique comportant l'activine a, l'alk-4 ou leurs derives pour le traitement des troubles ophtalmiques ou du cancer
WO2005094871A2 (fr) 2004-03-26 2005-10-13 Acceleron Pharma Inc. Propeptides bmp-3 et methodes associees
CA2561809A1 (fr) 2004-03-31 2005-10-20 Xencor, Inc. Variants de bmp-7 ayant des proprietes ameliorees
CA2572330A1 (fr) 2004-06-24 2006-01-05 Acceleron Pharma Inc. Propeptides gdf3 et methodes associees
ES2710048T3 (es) * 2004-07-23 2019-04-22 Acceleron Pharma Inc Anticuerpos antagonistas del receptor ActRII
CA2581896C (fr) 2004-09-29 2015-11-10 Mount Sinai School Of Medicine Of New York University Compositions et methodes modulant la fsh et le recepteur de la fsh, inhibant la resorption osseuse osteoclastique et les pertes osseuses de l'osteoporose
WO2006055689A2 (fr) 2004-11-16 2006-05-26 Avidia Research Institute Squelettes proteiques et leurs utilisations
NZ538097A (en) 2005-02-07 2006-07-28 Ovita Ltd Method and compositions for improving wound healing
ES2392096T3 (es) 2005-02-16 2012-12-04 The General Hospital Corporation Uso de antagonistas de bmp para regular el metabolismo del hierro mediado por hepcidina y tratar la deficiencia de hierro
EP1884235A1 (fr) * 2005-04-26 2008-02-06 Ajinomoto Co., Inc. Inducteur de differenciation des progeniteurs myeloerythroides
US8067562B2 (en) 2005-11-01 2011-11-29 Amgen Inc. Isolated nucleic acid molecule comprising the amino acid sequence of SEQ ID NO:1
BRPI0618947B1 (pt) 2005-11-23 2022-07-19 Acceleron Pharma Inc Polipeptídeo actriia que se liga à ativina, polinucleotídeo isolado, polinucleotídeo recombinante, métodopara preparar um polipeptídeo actriia que se liga à ativina, preparação farmacêutica e uso de uma proteína de fusão actriia-fc para promover o crescimento ósseo e inibir a ressorção óssea
AU2006321906C1 (en) 2005-12-06 2014-01-16 Amgen Inc. Uses of myostatin antagonists
JP4822562B2 (ja) 2006-01-20 2011-11-24 ベックマン コールター， インコーポレイテッド 低ヘモグロビン濃度細胞百分率および鉄欠乏の検出における使用方法
CN101489544A (zh) 2006-05-09 2009-07-22 海玛奎斯特医药公司 治疗血液病的方法
ATE547099T1 (de) 2006-07-21 2012-03-15 Lyne Lab Inc Flüssige zusammensetzungen aus calciumacetat
CL2007002567A1 (es) 2006-09-08 2008-02-01 Amgen Inc Proteinas aisladas de enlace a activina a humana.
US7547781B2 (en) 2006-09-11 2009-06-16 Curis, Inc. Quinazoline based EGFR inhibitors containing a zinc binding moiety
WO2008060139A1 (fr) 2006-11-17 2008-05-22 Erasmus University Medical Center Rotterdam Procédés destinés à contrôler la minéralisation d'une matrice extracellulaire, procédés thérapeutiques se fondant sur ces procédés et médicaments destinés à être utilisés dans le cadre de ces procédés
JP5415279B2 (ja) * 2006-12-18 2014-02-12 アクセルロン ファーマ， インコーポレイテッド アクチビン−ＡｃｔＲＩＩアンタゴニストおよび赤血球レベルを増加させるためのその使用
US8895016B2 (en) * 2006-12-18 2014-11-25 Acceleron Pharma, Inc. Antagonists of activin-actriia and uses for increasing red blood cell levels
AU2008211007B2 (en) 2007-02-01 2013-09-19 Acceleron Pharma Inc. Activin-ActRIIa antagonists and uses for treating or preventing breast cancer
TW201803890A (zh) 2007-02-02 2018-02-01 艾瑟勒朗法瑪公司 衍生自ＡｃｔＲＩＩＢ的變體與其用途
ES2756725T3 (es) 2007-02-09 2020-04-27 Acceleron Pharma Inc Composiciones farmacéuticas que comprenden antagonistas de Activina-ActRIIA
US7947646B2 (en) 2007-03-06 2011-05-24 Amgen Inc. Variant activin receptor polypeptides
JP2010529041A (ja) 2007-06-01 2010-08-26 ワイス・エルエルシー Ｂｍｐ−１０活性を調整する方法および組成物
JP2010535708A (ja) 2007-08-03 2010-11-25 ビオマリン アイジーエー リミテッド デュシェンヌ型筋ジストロフィーの治療のための薬物併用
GB0715087D0 (en) 2007-08-03 2007-09-12 Summit Corp Plc Drug combinations for the treatment of duchenne muscular dystrophy
CA2699936A1 (fr) 2007-09-18 2009-03-26 Acceleron Pharma Inc. Antagonistes de l'activine-actriia et utilisations pour reduire ou empecher la secretion de fsh
PE20091163A1 (es) 2007-11-01 2009-08-09 Wyeth Corp Anticuerpos para gdf8
WO2009137613A2 (fr) 2008-05-06 2009-11-12 Joslin Diabetes Center, Inc. Procédés et compositions pour induire une adipogenèse brune

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US20090047281A1 (en) 2009-02-19
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US20200031903A1 (en) 2020-01-30
NZ707292A (en) 2017-06-30
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CN105797135A (zh) 2016-07-27
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